1. Field of the Invention
This invention relates to a method and agent for the detection of antibodies to Treponema pallidum. 
2. Related Art
Treponema pallidum is the causative agent of syphilis. Diagnosis of infections with this pathogen is important not only when infection is suspected, but it is also of great significance to exclude syphilis among pregnant women and blood donors. In prenatal care, the purpose of such diagnosis is to prevent congenital syphilis, i.e. a transfer of syphilis to the neonate, and any consequent harm to the child. Negative serologic results for syphilis constitute one selection criterion for blood donors.
Syphilis is primarily diagnosed serologically, i.e. by the detection of antibodies to Treponema pallidum and/or cardiolipin. Specific IgM antibodies are detectable already 14 days after infection and IgG antibodies at the latest from 4 weeks after infection. Only before that time is direct detection of the causal agent from infected tissue of the primary lesion is another decisive diagnostic criterion.
Diagnosis by culture, as is conventional for many other bacterial pathogens, is not possible as Treponema pallidum has not hitherto successfully been cultured in vitro, with the exception of propagation in cell cultures (U.S. Pat. No. 5,264,360).
Serologic detection methods may be divided into three groups depending upon the nature of antibody detected:
1. Nontreponemal tests. Tests such as the cardiolipin microflocculation test (CMT), which is known in the English-speaking world as the Venereal Disease Research Laboratory Test (VDRL test), the rapid plasma reagin test (RPR test) and the cardiolipin complement binding reaction (cardiolipin CBR) are based on the detection of antibodies to cardiolipin. These tests reveal positive results 3-5 weeks after infection or approx. 7-10 days after appearance of the primary lesion. Sensitivity is 60 to 87% in the primary stage and may be as high as 100% in secondary syphilis. Sensitivity does, however, fall in the later stages of the disease, such that up to 30% of the late stages are no longer reactive. When the VDRL test is performed quantitatively, titre may be correlated to the activity of the disease. The disadvantage of this test is the large proportion of 0.3-0.9% of false positive test results when screening blood donors and the occurrence of false negative results in sera having an elevated titre due to the prozone phenomenon, which may be observed in 1-2% of cases in the VDRL test in secondary syphilis.
2. Treponema-specific tests. Antibodies to the endoflagellae of Treponema pallidum are formed as syphilis progresses. As a result of antigen relatedness, these antibodies also react with the endoflagellae of other species of treponema. The endoflagellae of Treponema phagedenis (biotype Reiter) have thus also been used as an antigen for the diagnosis of syphilis (Flagellum ELISA, R. V. W. van Eijk et al., Genitourin. Med. 62, 367-372 (1988)). In the flagellum ELISA, the cut off for a positive test result is a compromise between sensitivity and specificity, as a result of which 0.8% of results are false positives and 2.7% false negatives.
3. Treponema pallidum-specific tests. These tests detect antibodies which react with Treponema pallidum or antigen preparations from this pathogen. These test systems include the Treponema pallidum haemagglutination test (TPHA), the fluorescent Treponema pallidum antibody absorption test (FTA-ABS) and the Nelson test (Treponema pallidum immobilisation test, TPI) together with ELISA systems based on sonicate antigen. TPHA and FTA-ABS are generally used in diagnostics.
The Nelson test involves a microscopic assessment of the extent to which complement activating antibodies in the patient""s serum inhibit the mobility of Treponema pallidum. Due to its high cost, the Nelson test is used virtually exclusively for scientific investigations.
In TPHA, erythrocytes to which the Treponema pallidum sonicate antigen is bound are agglutinated by serum antibodies from syphilis patients. At a false positive rate of 0.07% and false negative rate of 0.008%, TPHA is highly specific and sensitive with most false negative results occurring in the initial stage of syphilis. TPHA is positive starting from the 4th week of infection, exhibits sensitivity of 64% to 87% in primary syphilis with an initially low titre (80-320) which, on transition to secondary syphilis, may rise to above 5000 at a sensitivity of 100%. As the disease progresses further, TPHA remains positive when the titre has a tendency to fall during the latent stage.
In FTA-ABS, indirect immunofluorescence microscopy is used to detect the binding of specific antibodies in the serum under investigation onto Treponema pallidum attached to a solid support via a fluorescent-labelled secondary antibody. Sensitivity of detection is 86% to 100% in primary syphilis, reaches 100% in secondary syphilis and 96% to 100% in the late stages. According to various sources, sensitivity for all stages is between 83% and 95%. Specificity, at 83% to 89% (excluding xe2x80x9cborderlinexe2x80x9d findings) is not very high. Isolated FTA-positive sera with an otherwise negative syphilis serology are caused, for example, by Lyme borreliosis. FTA-ABS is performed as a confirmatory test to validate positive TPHA findings. Veldkamp and Visser (Brit. J. Vener. Dis. 51, 227-231 (1975)) provide the first description of an ELISA (enzyme-linked immunosorbent assay) in which the sonicate antigen of Treponema pallidum, which is adsorptively bound to solid phases, reacts with specific antibodies from the material under investigation. The bound antibodies are detected by reaction with an enzyme-labelled, species-specific secondary antibody to class IgG or IgM immunoglobulins and a subsequent enzymatic colour reaction. The commercial indirect enzyme immunoassay Captia Syphilis G (Mercia Diagnostics, Guildford, England) is also based on this principle. This assay has been found to have a sensitivity of 98.4% and a specificity of 99.3% (Young et al., J. Clin. Pathol. 45, 37-41, (1992), Genitourin. Med. 65, 72-78 (1989)). Captia Syphilis M detects a specific IgM which is formed right at the early stages of the disease (sensitivity in primary syphilis 82%). Since IgM antibodies cannot pass through the placenta, this assay is of significance in the diagnosis of congenital syphilis (sensitivity 100%, Ijsselmuiden et al., J. Clin. Microbiol. 27, 152-157, (1989)). The assay is based on the principle of a solid phase bound capture antibody for IgM from the material under investigation, wherein specific IgM antibodies subsequently react with Treponema pallidum antigen. Detection is by means of an enzyme-labelled monoclonal antibody to endoflagellae and subsequent enzymatic colour reaction.
In the Syphilis BioEnza Bead Assay (Organon Teknika Corp., USA), the Treponema pallidum antigen is adsorptively bound to ferromagnetic particles. Binding of antibodies from the material under investigation is then detected by means of enzyme-labelled secondary antibodies and colour reaction. The sensitivity and specificity of this test are stated at 93% and 98.6% (Burdash et al., J. Clin. Microbiol. 25, 808-811, (1987)).
The sonicate antigen mentioned above is obtained by mechanical processing of the pathogen with ultrasound.
The deficient in vitro culturability of Treponema pallidum, which has already been mentioned, naturally means that these pathogens can be made available only with great difficulty.
Two-stage diagnosis of syphilis is currently generally recognised. In the first stage, all the sera are investigated with a test (screening). In the second stage, positive sera are investigated with another test system (confirmatory test). Since, in many countries, the incidence of syphilis is low ( less than 1%), 99% specificity of the screening test is not sufficient to ensure reliability of diagnosis. A positive predictive value of above 90% is only achieved with a confirmatory test based on a different detection principle.
Screening is widely performed using TPHA alone or in combination with the VDRL test or RPR test and the resultant positive sera are verified by FTA-ABS as the confirmatory test.
Performing the VDRL test or RPR test quantitatively is a recognised method for monitoring treatment, wherein a more than four-fold fall in titre is considered to indicate success of the treatment.
Valuation of current syphilis diagnostics: non-treponema-specific tests yield approx. 5% false positive results. They are thus suitable only as a first stage for serological diagnostics and, in the event of a positive result, must be complemented in the second stage by Treponema pallidum-specific tests. Since quantitative evaluation of non-treponema-specific tests may be correlated with the activity of the disease, such tests may be used to monitor the success of antibiotic treatment.
False positive test results in the treponema-specific tests are possible due to the relatedness of the antigens to treponema which occur in humans as commensal organisms in the oral mucosa. These test systems cannot gain general acceptance for diagnosing syphilis due to their deficient specificity and sensitivity.
Treponema pallidum-specific tests are based on Treponema pallidum antigen preparations, wherein, due to the deficient in vitro culturability of Treponema pallidum, the bacterial antigen used is obtained from infected rabbit testes. This method is associated with animal welfare considerations and only small quantities of antigen may be produced. The FTA-ABS and Nelson test are costly to perform and are not suitable for automation. Since antibodies to non-pathogenic treponema may also occur in healthy subjects and, due to relatedness of the antibodies, they exhibit cross-reactivity with Treponema pallidum sonicate antigen, the use of Treponema pallidum whole antigen requires serum absorption with Treponema phagedenis preparations.
While ELISA systems based on Treponema pallidum as a whole antigen do indeed satisfy quality criteria with regard to sensitivity and specificity, due to high cost, however,they are not suitable to replace the widely used two-stage diagnostics with TPHA, VDRL test or RPR test in combination with FTA-ABS.
It has already been established in EP 0 391 330 B1 that commercially available tests also do not fulfil the requirements placed on screening tests with regard to reasonable technical complexity and test evaluation. It is proposed in this document to remedy these defects by binding the antigens to a solid phase, either irreversibly directly or via a non-immunochemically binding spacer.
The object of the invention is accordingly to provide a method and agent for detecting antibodies to Treponema pallidum which allow this situation to be improved.
This object is achieved by a method in which a selection of recombinant antigens is gene-amplified and cloned, these gene-amplified and cloned antigens are subsequently expressed in host vector systems and then purified and the purified antigens are then bound to a solid phase individually or in combination, the antigens bound to the solid phase are then subjected to a reaction with a material to be investigated for antibodies, and determination of antibodies bound via antigen/antibody reaction qualitatively and/or quantitatively by means of a detection system.
Such a method for the first time provides a method which is reasonable with regard to technical complexity and simultaneously achieves the necessary maximum sensitivity and specificity when determining antibodies to Treponema pallidum. 
A means according to the invention of the method for detecting antibodies to Treponema pallidum consists of the recombinant antigens 17 kD antigen, 47 kD antigen and TmpA.
A combination of these three antigens results in an elevated diagnostic sensitivity in serological syphilis diagnostics. It allows the simultaneous detection of antibodies to antigens to which elevated antibody concentrations are to be found in all stages of syphilis.
In certain applications, it is preferred to add certain recombinant antigens either individually to the above-stated three or also in groups of two or more to the above-stated three. These added antigens are BMP, the 34 kD antigen, TmpC and Tp4.
Addition of these recombinant antigens is preferred in certain applications because additional detection of antibodies against Treponema pallidum-specific epitopes of these recombinant antigens may bring about a further improvement in sensitivity of the serological detection of syphilis.
The following preconditions apply to the optional addition of these recombinant antigens in order to avoid reductions in specificity:
1. non-specific binding must be minimised.
2. cross-reacting antibodies in the material under investigation must be removed or neutralised, for example by absorption.
When making combined use of recombinant antigens, it must be ensured for the particular embodiment that antibodies to each individual antigen generate a substantial proportion of the detected signal (increase in absorbance, chemiluminescence, radioactivity, fluorescence etc.).
While proposals have indeed already been made to clone Treponema pallidum DNA to overcome deficient in vitro culturability, no commercially usable diagnostic systems have come into being which are able to match the sensitivity and specificity of prior art syphilis diagnostics. Accordingly, only speculation is known from the literature, but no reports of serodiagnostic methods for syphilis using a combination of recombinant antigens.
It has been found that one fundamental problem associated with serum diagnostics using single antigens is that the level of antibodies for the individual antigens depend not only upon the stage of the disease, but also on idiosyncratic, individual differences. Combined antibody detection of two diagnostically relevant antigens, i.e. a selection thereof, may accordingly increase the sensitivity and specificity of the serological diagnostics.
Further, preferred means according to the invention are stated in the subordinate claims.
Another decisive factor for test systems using recombinant proteins is the purity thereof. When using recombinant antigens from expression systems with E. coli, it is necessary to remove the host proteins as completely as possible as natural antibodies to this intestinal bacterium are otherwise detected in the test system. The production of recombinant proteins using a cloning strategy, expression and purification of the recombinant proteins used, is thus of considerable significance to the particular test method.
The method according to the invention is based on the reaction of specific antibodies, in particular from body fluids such as serum, plasma or spinal fluid, with recombinant antigens of Treponema pallidum, which are bound to a solid phase. Antibodies bound by a specific antigen/antibody reaction are preferably detected by a secondary, labelled antibody. Labelling may be achieved by radioactive labels, fluorescent dyes or enzymes. The parameter measured is the bound radioactivity (principle of the radioimmunoassay), fluorescence (fluorescent immunoassay), enzyme activity by a substrate colour reaction (calorimetric immunoassay) or a substrate reaction with chemiluminescence (chemiluminescent immunoassay). In order to illustrate the described essentials of the invention by way of example, methods using colormetric and chemiluminescent detection are presented.
The antigens are bound to a solid phase using various principles. Possible binding principles are adsorptive binding, covalent binding or binding by reaction with a specific capture antibody already bound to a solid phase. Support materials which may be used are microparticles, beads, rods, tubes and microtest plates or porous materials. Test systems using microtest plates, to which the antigens have been adsorptively bound, and a detection method with the antigens covalently bound to paramagnetic particles are presented by way of example.
This method is used together with a selection of Treponema pallidum recombinant antigens. The genes for the 17 kD antigen, for the 47 kD antigen, TmpA, TmpC and BMP, the 34 kD antigen and Tp4 were amplified from Treponema pallidum DNA by polymerase chain reaction, inserted into the plasmid vector pQE-30 and transformed in E. coli M15[pREP4]. The host/vector system used allowed inducible expression of the recombinant proteins with subsequent purification of the recombinant proteins by affinity chromatography, the proteins then being bound to the solid phase.
The present invention furthermore provides the method for the selection of the recombinant antigens which are suitable for syphilis serodiagnostics by testing the antibody content of patient sera from different stages and the combined detection of antibodies to selected Treponema pallidum antigens.